MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Metagenomic next-generation sequencing to characterize potential etiologies of non-malarial fever in a cohort living in a high malaria burden area of Uganda.

Causes of non-malarial fevers in sub-Saharan Africa remain understudied. We hypothesized that metagenomic next-generation sequencing (mNGS), which allows for broad genomic-level detection of infectious agents in a biological sample, can systematically identify potential causes of non-malarial fevers. The 212 participants in this study were of all ages and were enrolled in a longitudinal malaria cohort in eastern Uganda. Between December 2020 and August 2021, respiratory swabs and plasma samples were collected at 313 study visits where participants presented with fever and were negative for malaria by microscopy. Samples were analyzed using CZ ID, a web-based platform for microbial detection in mNGS data. Overall, viral pathogens were detected at 123 of 313 visits (39%). SARS-CoV-2 was detected at 11 visits, from which full viral genomes were recovered from nine. Other prevalent viruses included Influenza A (14 visits), RSV (12 visits), and three of the four strains of seasonal coronaviruses (6 visits). Notably, 11 influenza cases occurred between May and July 2021, coinciding with when the Delta variant of SARS-CoV-2 was circulating in this population. The primary limitation of this study is that we were unable to estimate the contribution of bacterial microbes to non-malarial fevers, due to the difficulty of distinguishing bacterial microbes that were pathogenic from those that were commensal or contaminants. These results revealed the co-circulation of multiple viral pathogens likely associated with fever in the cohort during this time period. This study illustrates the utility of mNGS in elucidating the multiple potential causes of non-malarial febrile illness. A better understanding of the pathogen landscape in different settings and age groups could aid in informing diagnostics, case management, and public health surveillance systems.

Metagenomic Pathogen Sequencing in Resource-Scarce Settings: Lessons Learned and the Road Ahead

Metagenomic next-generation sequencing (mNGS) is the process of sequencing all genetic material in a biological sample. The technique is growing in popularity with myriad applications including outbreak investigation, biosurveillance, and pathogen detection in clinical samples. However, mNGS programs are costly to build and maintain, and additional obstacles faced by low- and middle-income countries (LMICs) may further widen global inequities in mNGS capacity. Over the past two decades, several important infectious disease outbreaks have highlighted the importance of establishing widespread sequencing capacity to support rapid disease detection and containment at the source. Using lessons learned from the COVID-19 pandemic, LMICs can leverage current momentum to design and build sustainable mNGS programs, which would form part of a global surveillance network crucial to the elimination of infectious diseases.

Using Split Luminescent Biosensors for SARS-CoV-2 Antibody Detection in Serum, Plasma, and Blood Samples.

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days.

Full Genome Nobecovirus Sequences From Malagasy Fruit Bats Define a Unique Evolutionary History for This Coronavirus Clade.

Bats are natural reservoirs for both Alpha- and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat (Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis. Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent ancestral sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.

IDseq-An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource-limited environments. FINDINGS: We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline, which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics that are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. CONCLUSION: The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

The COVID-19 epidemic in Madagascar: clinical description and laboratory results of the first wave, march-september 2020.

BACKGROUND: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country. METHODS: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PCR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory-confirmed cases were mapped, and six genomes of viruses were fully sequenced. RESULTS: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2-positive patients, the median age was 39 years (IQR: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A and 20B indicative of several independent introduction of viruses. CONCLUSIONS: Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave.

SARS-CoV-2 vaccines in advanced clinical trials: Where do we stand?

The ongoing SARS-CoV-2 pandemic has led to the focused application of resources and scientific expertise toward the goal of developing investigational vaccines to prevent COVID-19. The highly collaborative global efforts by private industry, governments and non-governmental organizations have resulted in a number of SARS-CoV-2 vaccine candidates moving to Phase III trials in a period of only months since the start of the pandemic. In this review, we provide an overview of the preclinical and clinical data on SARS-CoV-2 vaccines that are currently in Phase III clinical trials and in few cases authorized for emergency use. We further discuss relevant vaccine platforms and provide a discussion of SARS-CoV-2 antigens that may be targeted to increase the breadth and durability of vaccine responses.

Complete Genome Sequence of a Novel Coronavirus (SARS-CoV-2) Isolate from Bangladesh.

The complete genome sequence of a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) isolate obtained from a nasopharyngeal swab from a patient with COVID-19 in Bangladesh is reported.